Fibronectin fibrillogenesis is an integrin-mediated process that may contribute to the pathogenesis of primary open-angle glaucoma (POAG). Here, we examined the effects of αvβ3 integrins on fibrillogenesis in immortalized TM-1 cells and human trabecular meshwork (HTM) cells.
TM-1 cells overexpressing wild-type β3 (WTβ3) or constitutively active β3 (CAβ3) integrin subunits were generated. Control cells were transduced with an empty vector (EV). Deoxycholic acid (DOC) extraction of monolayers, immunofluorescence microscopy, and On-cell western analyses were used to determine levels of fibronectin fibrillogenesis and fibronectin fibril composition (EDA+ and EDB+ fibronectins) and conformation. αvβ3 and α5β1 Integrin levels were determined using fluorescence-activated cell sorting (FACS). Cilengitide and an adenovirus vector expressing WTβ3 or CAβ3 integrin subunits were used to examine the role of αvβ3 integrin in HTM cells. The role of the canonical α5β1 integrin-mediated pathway in fibrillogenesis was determined using the fibronectin-binding peptide FUD, the β1 integrin function-blocking antibody 13, and the Rho kinase (ROCK) inhibitor Y27632.
Activation of αvβ3 integrin enhanced the assembly of fibronectin into DOC-insoluble fibrils in both TM-1 and HTM cells. The formation of fibronectin fibrils was dependent on α5β1 integrin and could be inhibited by FUD. However, fibrillogenesis was unaffected by Y27632. Fibrils assembled by CAβ3 cells also contained high levels of EDA+ and EDB+ fibronectin and fibronectin that was stretched.
αvβ3 Integrin signaling altered the deposition and structure of fibronectin fibrils using a β1 integrin/ROCK-independent mechanism. Thus, αvβ3 integrins could play a significant role in altering the function of fibronectin matrices in POAG.

In many cancer types, integrin-mediated signaling regulates proliferation, survival and invasion of tumorigenic cells. However, it is still unclear how integrins crosstalk with oncogenes to regulate tumorigenesis and metastasis. Here we show that oncogenic K-RasV12 upregulates α6-integrin expression in Madin-Darby canine kidney (MDCK) cells via activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)/Fos-related antigen 1-signaling cascade. Activated α6-integrins promoted metastatic capacity and anoikis resistance, and led to perturbed growth of MDCK cysts. Transcriptomic analysis of K-RasV12-transformed MDCK cells also revealed robust downregulation of αV-class integrins. Re-expression of αV-integrin in K-RasV12-transformed MDCK cells synergistically upregulated the expression of Zinc finger E-box-binding homeobox 1 and Twist-related protein 1 and triggered epithelial-mesenchymal transition leading to induced cell motility and invasion. These results delineate the signaling cascades connecting oncogenic K-RasV12 with α6- and αV-integrin functions to modulate cancer cell survival and tumorigenesis, and reveal new possible strategies to target highly oncogenic K-RasV12 mutants.

Estrogen receptor α positive (ERα+) breast cancer accounts for most breast cancer deaths. Both prolactin (PRL) and extracellular matrix (ECM) stiffness/density have been implicated in metastatic progression of this disease. We previously demonstrated that these factors cooperate to fuel processes involved in cancer progression. Culture of ERα+ breast cancer cells in dense/stiff 3D collagen-I matrices shifts the repertoire of PRL signals, and increases crosstalk between PRL and estrogen to promote proliferation and invasion. However, previous work did not distinguish ECM stiffness and collagen density. In order to dissect the ECM features that control PRL signals, we cultured T47D and MCF-7 cells on polyacrylamide hydrogels of varying elastic moduli (stiffness) with varying collagen-I concentrations (ligand density). Increasing stiffness from physiological to pathological significantly augmented PRL-induced phosphorylation of ERK1/2 and the SFK target, FAK-Y925, with only modest effects on pSTAT5. In contrast, higher collagen-I ligand density lowered PRL-induced pSTAT5 with no effect on pERK1/2 or pFAK-Y925. Disrupting focal adhesion signaling decreased PRL signals and PRL/estrogen-induced proliferation more efficiently in stiff, compared to compliant, extracellular environments. These data indicate that matrix stiffness shifts the balance of PRL signals from physiological (JAK2/STAT5) to pathological (FAK/SFK/ERK1/2) by increasing PRL signals through focal adhesions. Together, our studies suggest that PRL signaling to FAK and SFKs may be useful targets in clinical aggressive ERα+ breast carcinomas.

Clinically, circulating prolactin levels and density of the extracellular matrix (ECM) are individual risk factors for breast cancer. As tumors develop, the surrounding stroma responds with increased deposition and cross-linking of the collagen matrix (desmoplasia). In mouse models, prolactin promotes mammary carcinomas that resemble luminal breast cancers in women, and increased collagen density promotes tumor metastasis and progression. Although the contributions of the ECM to the physiologic actions of prolactin are increasingly understood, little is known about the functional relationship between the ECM and prolactin signaling in breast cancer. Here, we examined consequences of increased ECM stiffness on prolactin signals to luminal breast cancer cells in three-dimensional collagen I matrices in vitro. We showed that matrix stiffness potently regulates a switch in prolactin signals from physiologic to protumorigenic outcomes. Compliant matrices promoted physiological prolactin actions and activation of STAT5, whereas stiff matrices promoted protumorigenic outcomes, including increased matrix metalloproteinase-dependent invasion and collagen scaffold realignment. In stiff matrices, prolactin increased SRC family kinase-dependent phosphorylation of focal adhesion kinase (FAK) at tyrosine 925, FAK association with the mitogen-activated protein kinase mediator GRB2, and pERK1/2. Stiff matrices also increased co-localization of prolactin receptors and integrin-activated FAK, implicating altered spatial relationships. Together, these results demonstrate that ECM stiffness is a powerful regulator of the spectrum of prolactin signals and that stiff matrices and prolactin interact in a feed-forward loop in breast cancer progression. Our study is the first reported evidence of altered ECM-prolactin interactions in breast cancer, suggesting the potential for new therapeutic approaches.

We describe the generation of monocyte-derived dendritic cells (MoDC) from leukapheresis products using the CliniMACS system from Miltenyi Biotec. In a clinical setting, the method turned out to be feasible for the generation of clinical-grade MoDC from patients with metastatic renal-cell carcinoma. MoDC generated with this system exhibited a fully mature phenotype as well as high migratory and T-cell stimulatory capacity.